Project Summary Temperature control technology is necessary for a broad range of biologically relevant processes including organ-on-chip operation, biomolecular kinetics, cell growth, studying gene function with temperature-sensitive mutations, cancer cell resistance to hyperthermia treatments, protein crystallization, and DNA analysis. Most biosensing devices lack the needed temperature measurement accuracy and precise temperature control to understand the thermal mechanisms of these processes. For example, temperature variations of 0.2C can activate heat shock proteins, increasing the resistance of cancer cells to thermal ablation treatment, but reported temperature accuracies are often near 1C. This proposal aims to revolutionize the biomedical temperature measurement and control ecosystem by developing technology, models, and validated devices capable of microscopic, spatially resolved temperature sensing and control at 0.1C accuracy (10x better than what is used in most biosensing systems). Microfluidics is a promising technology for an extremely broad range of biomedical applications that notably lacks the necessary temperature accuracies and spatial temperature control to effectively study biothermal mechanisms. This proposal intends to impact human health by developing disruptive temperature control tools to accelerate biomedical innovation in thermally sensitive processes. Our group recently demonstrated the capacity to measure temperature at a single point with fluorescent dyes, achieving a 0.05C noise floor by using machine learning techniques. We have also 3D printed a cell-based genotype and phenotype assay device with cell growth chambers, monoliths for mRNA capture & fluorescence measurement, and integrated pumps and valves in a volume of only 2.2 mm 2.2 mm 1 mm. Aims 1 and 2 of this proposal will build on these successes by developing 3D printing technologies that easily incorporate complex temperature sensing, heating, and cooling channels, coupled with multi-physics/CAD models to rapidly iterate through the prototype development cycle. These advances will be used in Aim 3 to construct a microscopically temperature-controlled chip to measure DNA melt curves to determine the zygosity of a Factor 5 Leiden. This will show that the technology can detect the subtle difference in melting temperature that is undetectable by most PCR machines, as a proof-of-concept before the technology can be applied to other biological process. The overall objective of these studies is to develop a suite of affordable technologies researchers can use to understand biothermal mechanisms to lay the foundation for advances in disease diagnosis, treatment, and prevention.